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Temperate P335 phage TP901-1 represents one of the best-characterized Gram-positive phages regarding its structure and host interactions. Following its reversible adsorption to the polysaccharidic side-chain of the cell wall polysaccharide of its host Lactococcus cremoris 3107, TP901-1 requires a glucosylated cell envelope moiety to trigger its genome delivery into the host cytoplasm. Here, we demonstrate that three distinct single amino acid substitutions in the Tal protein of TP901-1 baseplate are sufficient to overcome the TP901-1 resistance of three L. cremoris 3107 derivatives, whose resistance is due to impaired DNA release of the phage. All of these Tal alterations are located in the N-terminally located gp27-like domain of the protein, conserved in many tailed phages. AlphaFold2 predictions of the Tal mutant proteins suggest that these mutations favor conformational changes necessary to reposition the Tal fiber and thus facilitate release of the tape measure protein from the tail tube and subsequent DNA ejection in the absence of the trigger otherwise required for phage genome release. IMPORTANCE: Understanding the molecular mechanisms involved in phage-host interactions is essential to develop phage-based applications in the food and probiotic industries, yet also to reduce the risk of phage infections in fermentations. Lactococcus, extensively used in dairy fermentations, has been widely employed to unravel such interactions. Phage infection commences with the recognition of a suitable host followed by the release of its DNA into the bacterial cytoplasm. Details on this latter, irreversible step are still very scarce in lactococci and other Gram-positive bacteria. We demonstrate that a component of the baseplate of the lactococcal phage TP901-1, the tail-associated lysin (Tal), is involved in the DNA delivery into its host, L. cremoris 3107. Specifically, we have found that three amino acid changes in Tal appear to facilitate structural rearrangements in the baseplate necessary for the DNA release process, even in the absence of an otherwise required host trigger.
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Until the late 2000s, lactococci substantially contributed to the discovery of various plasmid-borne phage defence systems, rendering these bacteria an excellent antiphage discovery resource. Recently, there has been a resurgence of interest in identifying novel antiphage systems in lactic acid bacteria owing to recent reports of so-called 'defence islands' in diverse bacterial genera. Here, 321 plasmid sequences from 53 lactococcal strains were scrutinized for the presence of antiphage systems. Systematic evaluation of 198 candidates facilitated the discovery of seven not previously described antiphage systems, as well as five systems, of which homologues had been described in other bacteria. All described systems confer resistance against the most prevalent lactococcal phages, and act post phage DNA injection, while all except one behave like abortive infection systems. Structure and domain predictions provided insights into their mechanism of action and allow grouping of several genetically distinct systems. Although rare within our plasmid collection, homologues of the seven novel systems appear to be widespread among bacteria. This study highlights plasmids as a rich repository of as yet undiscovered antiphage systems.
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The biosynthetic machinery for cell wall polysaccharide (CWPS) formation in Lactococcus lactis and Lactococcus cremoris is encoded by the cwps locus. The CWPS of lactococci typically consists of a neutral rhamnan component, which is embedded in the peptidoglycan, and to which a surface-exposed side chain oligosaccharide or polysaccharide pellicle (PSP) component is attached. The rhamnan component has been shown for several lactococcal strains to consist of a repeating rhamnose trisaccharide subunit, while the side chain is diverse in glycan content, polymeric status and glycosidic linkage architecture. The observed structural diversity of the CWPS side chain among lactococcal strains is reflected in the genetic diversity within the variable 3' region of the corresponding cwps loci. To date, four distinct cwps genotypes (A, B, C, D) have been identified, while eight subtypes (C1 through to C8) have been recognized among C-genotype strains. In the present study, we report the identification of three novel subtypes of the lactococcal cwps C genotypes, named C9, C10 and C11. The CWPS of four isolates representing C7, C9, C10 and C11 genotypes were analysed using 2D NMR to reveal their unique CWPS structures. Through this analysis, the structure of one novel rhamnan, three distinct PSPs and three exopolysaccharides were elucidated. Results obtained in this study provide further insights into the complex nature and fascinating diversity of lactococcal CWPSs. This highlights the need for a holistic view of cell wall-associated glycan structures which may contribute to robustness of certain strains against infecting bacteriophages. This has clear implications for the fermented food industry that relies on the consistent application of lactococcal strains in mesophilic production systems.
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Pared Celular , Genotipo , Lactococcus , Leche , Pared Celular/química , Lactococcus/genética , Lactococcus/aislamiento & purificación , Lactococcus/clasificación , Animales , Leche/microbiología , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/metabolismo , Alimentos Fermentados/microbiología , Polisacáridos/metabolismo , Microbiología de AlimentosRESUMEN
The persistent challenge of phages in dairy fermentations requires the development of starter cultures with enhanced phage resistance. Recently, three plasmid-encoded lactococcal antiphage systems, named Rhea, Aristaios, and Kamadhenu, were discovered. These systems were found to confer high levels of resistance against various Skunavirus members. In the present study, their effectiveness against phage infection was confirmed in milk-based medium, thus validating their potential to ensure reliable dairy fermentations. We furthermore demonstrated that Rhea and Kamadhenu do not directly hinder phage genome replication, transcription, or associated translation. Conversely, Aristaios was found to interfere with phage transcription. Two of the antiphage systems are encoded on pMRC01-like conjugative plasmids, and the Kamadhenu-encoding plasmid was successfully transferred by conjugation to three lactococcal strains, each of which acquired substantially enhanced phage resistance against Skunavirus members. Such advances in our knowledge of the lactococcal phage resistome and the possibility of mobilizing these protective functions to bolster phage protection in sensitive strains provide practical solutions to the ongoing phage problem in industrial food fermentations.IMPORTANCEIn the current study, we characterized and evaluated the mechanistic diversity of three recently described, plasmid-encoded lactococcal antiphage systems. These systems were found to confer high resistance against many members of the most prevalent and problematic lactococcal phage genus, rendering them of particular interest to the dairy industry, where persistent phage challenge requires the development of starter cultures with enhanced phage resistance characteristics. Our acquired knowledge highlights that enhanced understanding of lactococcal phage resistance systems and their encoding plasmids can provide rational and effective solutions to the enduring issue of phage infections in dairy fermentation facilities.
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Commercially produced cyanobacteria preparations sold under the name spirulina are widely consumed, due to their traditional use as a nutrient-rich foodstuff and subsequent marketing as a superfood. Despite their popularity, the microbial composition of ponds used to cultivate these bacteria is understudied. A total of 19 pond samples were obtained from small-scale spirulina farms and subjected to metagenome and/or virome sequencing, and the results were analysed. A remarkable level of prokaryotic and viral diversity was found to be present in the ponds, with Limnospira sp. and Arthrospira sp. sometimes being notably scarce. A detailed breakdown of prokaryotic and viral components of 15 samples is presented. Twenty putative Limnospira sp.-infecting bacteriophage contigs were identified, though no correlation between the performance of these cultures and the presence of phages was found. The high diversity of these samples prevented the identification of clear trends in sample performance over time, between ponds or when comparing successful and failed fermentations.
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Bacteriófagos , Biodiversidad , Fermentación , Metagenómica , Spirulina , Metagenómica/métodos , Spirulina/genética , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Bacteriófagos/clasificación , Metagenoma , Viroma , Filogenia , Estanques/microbiología , Estanques/virología , Bacterias/virología , Bacterias/genética , Bacterias/clasificación , Bacterias/aislamiento & purificaciónRESUMEN
Plasmids pNP40 and pUC11B encode two prevalent yet divergent conjugation systems, which have been characterized in detail recently. Here, we report the elucidation of the putative adhesins of the pNP40 and pUC11B conjugation systems, encoded by traAd and trsAd, respectively. Despite their significant sequence divergence, TraAd and TrsAd represent the most conserved component between the pNP40- and the pUC11B-encoded conjugation systems and share similar peptidoglycan-hydrolase domains. Protein structure prediction using AlphaFold2 highlighted the structural similarities between their predicted domains, as well as the potential homo-dimeric state of both proteins. Expression of the putative surface adhesins resulted in a cell clumping phenotype not only among cells expressing these surface adhesins but also between adhesin-expressing and non-producing cells. Furthermore, mutant derivatives of plasmids pNP40 or pUC11B carrying a mutation in traAd or trsAd, respectively, were shown to act as efficient donors provided the corresponding recipient expresses either traAd or trsAd, thus demonstrating in trans reciprocal complementarity of these proteins in conjugation systems.
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The distinct conjugation machineries encoded by plasmids pNP40 and pUC11B represent the most prevalent plasmid transfer systems among lactococcal strains. In the current study, we identified genetic determinants that underpin pNP40- and pUC11B-mediated, high-frequency mobilisation of other, non-conjugative plasmids. The mobilisation frequencies of the smaller, non-conjugative plasmids and the minimal sequences required for their mobilisation were determined, owing to the determination of the oriT sequences of both pNP40 and pUC11B, which allowed the identification of similar sequences in some of the non-conjugative plasmids that were shown to promote their mobilisation. Furthermore, the auxiliary gene mobC, two distinct functional homologues of which are present in several plasmids harboured by the pNP40- and pUC11B-carrying host strains, was observed to confer a high-frequency mobilisation phenotype. These findings provide mechanistic insights into how lactococcal conjugative plasmids achieve conjugation and promote mobilisation of non-conjugative plasmids. Ultimately, these insights would be harnessed to optimise conjugation and mobilisation strategies for the rapid and predictable development of robust and technologically improved strains.
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Conjugación Genética , Transferencia de Gen Horizontal , Plásmidos , Plásmidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Lactococcus lactis/genéticaRESUMEN
Bacteria have evolved diverse defense mechanisms to counter bacteriophage attacks. Genetic programs activated upon infection characterize phage-host molecular interactions and ultimately determine the outcome of the infection. In this study, we applied ribosome profiling to monitor protein synthesis during the early stages of sk1 bacteriophage infection in Lactococcus cremoris. Our analysis revealed major changes in gene expression within 5 minutes of sk1 infection. Notably, we observed a specific and severe downregulation of several pyr operons which encode enzymes required for uridine monophosphate biosynthesis. Consistent with previous findings, this is likely an attempt of the host to starve the phage of nucleotides it requires for propagation. We also observed a gene expression response that we expect to benefit the phage. This included the upregulation of 40 ribosome proteins that likely increased the host's translational capacity, concurrent with a downregulation of genes that promote translational fidelity (lepA and raiA). In addition to the characterization of host-phage gene expression responses, the obtained ribosome profiling data enabled us to identify two putative recoding events as well as dozens of loci currently annotated as pseudogenes that are actively translated. Furthermore, our study elucidated alterations in the dynamics of the translation process, as indicated by time-dependent changes in the metagene profile, suggesting global shifts in translation rates upon infection. Additionally, we observed consistent modifications in the ribosome profiles of individual genes, which were apparent as early as 2 minutes post-infection. The study emphasizes our ability to capture rapid alterations of gene expression during phage infection through ribosome profiling. IMPORTANCE: The ribosome profiling technology has provided invaluable insights for understanding cellular translation and eukaryotic viral infections. However, its potential for investigating host-phage interactions remains largely untapped. Here, we applied ribosome profiling to Lactococcus cremoris cultures infected with sk1, a major infectious agent in dairy fermentation processes. This revealed a profound downregulation of genes involved in pyrimidine nucleotide synthesis at an early stage of phage infection, suggesting an anti-phage program aimed at restricting nucleotide availability and, consequently, phage propagation. This is consistent with recent findings and contributes to our growing appreciation for the role of nucleotide limitation as an anti-viral strategy. In addition to capturing rapid alterations in gene expression levels, we identified translation occurring outside annotated regions, as well as signatures of non-standard translation mechanisms. The gene profiles revealed specific changes in ribosomal densities upon infection, reflecting alterations in the dynamics of the translation process.
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Bacteriófagos , Lactococcus , Biosíntesis de Proteínas , Perfilado de Ribosomas , Regulación hacia Abajo , Bacteriófagos/genética , Bacteriófagos/metabolismo , ARN Mensajero/metabolismo , Nucleótidos/metabolismo , Uridina Monofosfato/metabolismoRESUMEN
Lactococcal conjugative plasmids are poorly characterized compared to those harbored by numerous other Gram-positive bacteria, despite their significance in dairy fermentations and starter culture development. Furthermore, the transcriptional landscape of these lactococcal conjugation systems and their regulation have not been studied in any detail. Lactococcal plasmids pNP40 and pUC11B possess two genetically distinct and prevalent conjugation systems. Here, we describe the detailed transcriptional analysis of the pNP40 and pUC11B conjugation-associated gene clusters, revealing three and five promoters, respectively, for which the corresponding transcriptional start sites were identified. Regulation of several of these promoters, and therefore conjugation, is shown to involve the individual or concerted activities of the corresponding relaxase and transcriptional repressor(s) encoded by each conjugative plasmid. This work highlights how the conjugative potential of these systems may be unlocked, with significant implications for the starter culture and food fermentation industry.
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The dairy fermentation industry relies on the activity of lactic acid bacteria in robust starter cultures to accomplish milk acidification. Maintenance of the composition of these starter cultures, whether defined or undefined, is essential to ensure consistent and high-quality fermentation end products. To date, limited information exists regarding the microbial composition of undefined starter culture systems. Here, we describe a culture-based analysis combined with a metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, we describe a qPCR-based genotype detection assay, which is capable of discerning nine distinct lactococcal genotypes to characterize these undefined starter cultures, and which can be applied to monitor compositional changes in an undefined starter culture during a fermentation. IMPORTANCE: This study reports on the development of a combined culture-based analysis and metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, a novel qPCR-based genotype detection assay, capable of discerning nine distinct lactococcal genotypes (based on lactococcal cell wall polysaccharide biosynthesis gene clusters), was used to monitor compositional changes in an undefined starter culture following phage attack. These analytical approaches facilitate a multifaceted assessment of starter culture compositional stability during milk fermentation, which has become an important QC aspect due to the increasing demand for consistent and high-quality dairy products.
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Bacteriófagos , Lactobacillales , Lactococcus lactis , Animales , Lactococcus lactis/genética , Leche/microbiología , Bacteriófagos/genética , FermentaciónRESUMEN
The field of microbial ecology has been transformed by metagenomics in recent decades and has culminated in vast datasets that facilitate the bioinformatic dissection of complex microbial communities. Recently, attention has turned from defining the microbiota composition to the interactions and relationships that occur between members of the microbiota. Within complex microbiota, the identification of bacteriophage-host combinations has been a major challenge. Recent developments in artificial intelligence tools to predict protein structure and function as well as the relationships between bacteria and their infecting bacteriophages allow a strategic approach to identifying and validating phage-host relationships. However, biological validation of these predictions remains essential and will serve to improve the existing predictive tools. In this review, I provide an overview of the most recent developments in both bioinformatic and experimental approaches to predicting and experimentally validating unknown phage-host combinations.
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Bacteriófagos , Microbiota , Bacteriófagos/genética , Inteligencia Artificial , Biología Computacional , MetagenómicaRESUMEN
In Gram-positive bacteria, cell wall polysaccharides (CWPS) play critical roles in bacterial cell wall homeostasis and bacterial interactions with their immediate surroundings. In lactococci, CWPS consist of two components: a conserved rhamnan embedded in the peptidoglycan layer and a surface-exposed polysaccharide pellicle (PSP), which are linked together to form a large rhamnose-rich CWPS (Rha-CWPS). PSP, whose structure varies from strain to strain, is a receptor for many bacteriophages infecting lactococci. Here, we examined the first two steps of PSP biosynthesis, using in vitro enzymatic tests with lipid acceptor substrates combined with LC-MS analysis, AlfaFold2 modeling of protein 3D-structure, complementation experiments, and phage assays. We show that the PSP repeat unit is assembled on an undecaprenyl-monophosphate (C55P) lipid intermediate. Synthesis is initiated by the WpsA/WpsB complex with GlcNAc-P-C55 synthase activity and the PSP precursor GlcNAc-P-C55 is then elongated by specific glycosyltransferases that vary among lactococcal strains, resulting in PSPs with diverse structures. Also, we engineered the PSP biosynthesis pathway in lactococci to obtain a chimeric PSP structure, confirming the predicted glycosyltransferase specificities. This enabled us to highlight the importance of a single sugar residue of the PSP repeat unit in phage recognition. In conclusion, our results support a novel pathway for PSP biosynthesis on a lipid-monophosphate intermediate as an extracellular modification of rhamnan, unveiling an assembly machinery for complex Rha-CWPS with structural diversity in lactococci.
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Pared Celular , Lactococcus , Polisacáridos Bacterianos , Ramnosa , Proteínas Bacterianas/metabolismo , Pared Celular/química , Pared Celular/metabolismo , Glicosiltransferasas/metabolismo , Lactococcus/clasificación , Lactococcus/citología , Lactococcus/metabolismo , Lactococcus/virología , Lípidos , Peptidoglicano/metabolismo , Polisacáridos Bacterianos/metabolismo , Conformación Proteica , Ramnosa/metabolismo , Especificidad por Sustrato , Bacteriófagos/fisiologíaRESUMEN
Bacteria are engaged in a constant battle against preying viruses, called bacteriophages (or phages). These remarkable nano-machines pack and store their genomes in a capsid and inject it into the cytoplasm of their bacterial prey following specific adhesion to the host cell surface. Tailed phages possessing dsDNA genomes are the most abundant phages in the bacterial virosphere, particularly those with long, non-contractile tails. All tailed phages possess a nano-device at their tail tip that specifically recognizes and adheres to a suitable host cell surface receptor, being proteinaceous and/or saccharidic. Adhesion devices of tailed phages infecting Gram-positive bacteria are highly diverse and, for the majority, remain poorly understood. Their long, flexible, multi-domain-encompassing tail limits experimental approaches to determine their complete structure. We have previously shown that the recently developed protein structure prediction program AlphaFold2 can overcome this limitation by predicting the structures of phage adhesion devices with confidence. Here, we extend this approach and employ AlphaFold2 to determine the structure of a complete phage, the lactococcal P335 phage TP901-1. Herein we report the structures of its capsid and neck, its extended tail, and the complete adhesion device, the baseplate, which was previously partially determined using X-ray crystallography.
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Bacteriófagos , Lactococcus lactis , Siphoviridae , Siphoviridae/genética , Bacteriófagos/genética , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Cristalografía por Rayos XRESUMEN
The Streptococcus genus comprises both commensal and pathogenic species. Additionally, Streptococcus thermophilus is exploited in fermented foods and in probiotic preparations. The ecological and metabolic diversity of members of this genus is matched by the complex range of cell wall polysaccharides that they present on their cell surfaces. These glycopolymers facilitate their interactions and environmental adaptation. Here, current knowledge on the genetic and compositional diversity of streptococcal cell wall polysaccharides including rhamnose-glucose polysaccharides, exopolysaccharides and teichoic acids is discussed. Furthermore, the species-specific cell wall polysaccharide combinations and specifically highlighting the presence of rhamnose-glucose polysaccharides in certain species, which are replaced by teichoic acids in other species. This review highlights model pathogenic and non-pathogenic species for which there is considerable information regarding cell wall polysaccharide composition, structure and genetic information. These serve as foundations to predict and focus research efforts in other streptococcal species for which such data currently does not exist.
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Ramnosa , Ácidos Teicoicos , Ácidos Teicoicos/análisis , Ramnosa/análisis , Ramnosa/metabolismo , Polisacáridos/química , Streptococcus/genética , Streptococcus/química , Streptococcus/metabolismo , Polisacáridos Bacterianos/genética , Polisacáridos Bacterianos/análisis , Polisacáridos Bacterianos/metabolismo , Pared Celular/metabolismo , GlucosaRESUMEN
Lactococcus spp. are applied routinely in dairy fermentations and their consistent growth and associated acidification activity is critical to ensure the quality and safety of fermented dairy foods. Bacteriophages pose a significant threat to such fermentations and thus it is imperative to study how these bacteria may evade their viral predators in the relevant confined settings. Many lactococcal phages are known to specifically recognise and bind to cell wall polysaccharides (CWPSs) and particularly the phospho-polysaccharide (PSP) side chain component that is exposed on the host cell surface. In the present study, we generated derivatives of a lactococcal strain with reduced phage sensitivity to establish the mode of phage evasion. The resulting mutants were characterized using a combination of comparative genome analysis, microbiological and chemical analyses. Using these approaches, it was established that the phage-resistant derivatives incorporated mutations in genes within the cluster associated with CWPS biosynthesis resulting in growth and morphological defects that could revert when the selective pressure of phages was removed. Furthermore, the cell wall extracts of selected mutants revealed that the phage-resistant strains produced intact PSP but in significantly reduced amounts. The reduced availability of the PSP and the ability of lactococcal strains to revert rapidly to wild type growth and activity in the absence of phage pressure provides Lactococcus with the means to survive and evade phage attack.
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Bacteriófagos , Lactococcus lactis , Bacteriófagos/genética , Bacteriófagos/metabolismo , Lactococcus lactis/metabolismo , Polisacáridos/análisis , Polisacáridos/química , Polisacáridos/metabolismo , Pared Celular/metabolismo , MutaciónRESUMEN
Lactococcus is one of the earliest identified fermentative bacterial genera and among its member species, the dairy-associated Lactococcus lactis and Lactococcus cremoris are undoubtedly the best studied. These two species are believed to have evolved from plant-associated lactococci and through genome decay and acquisition of plasmids, have adapted to the dairy niche. The past decade has witnessed a surge of activity in novel lactococcal species identification from insect, plant and animal sources. Currently, 22 Lactococcus species are described and in this review, we summarise the genome characteristics of and phylogenetic relationships among these species. Furthermore, we explore the role of mobile elements including plasmids and bacteriophages in the diversification of lactococcal species. The pace of identification of novel lactococcal species suggests that the number of lactococcal species is likely to continue to grow. With additional sequence data for the emerging species, it will be possible to perform pathogenicity/virulence risk evaluations and generate extensive insights into the niche adaptation strategies through which they have evolved.
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Bacteriófagos , Lactococcus lactis , Animales , Filogenia , Lactococcus lactis/genética , Bacteriófagos/genética , Fermentación , VirulenciaRESUMEN
Lactococcus cremoris and Lactococcus lactis are among the most extensively exploited species of lactic acid bacteria in dairy fermentations. The cell wall of lactococci, like other Gram-positive bacteria, possesses a thick peptidoglycan layer, which may incorporate cell wall polysaccharides (CWPS), wall teichoic acids (WTA), and/or lipoteichoic acids (LTA). In this study, we report the isolation, purification and structural analysis of the carbohydrate moieties of glycolipids (GL) and LTA of the L. cremoris model strain 3107. Chemical structures of these compounds were studied by chemical methods, NMR spectroscopy and positive and negative mode ESI MS. We found that the LTA of strain 3107 is composed of short chains of 1,3-polyglycerol phosphate (PGP), attached to O-6 of the non-reducing glucose of the kojibiose-Gro backbone of the glycolipid anchor. Extraction of cells with cold TCA afforded the detection of 1,3-glycerol phosphate chains randomly substituted at O-2 of glycerol by D-Ala. Unlike the LTA of L. lactis strains studied to date, the PGP backbone of the LTA of L. cremoris 3107 did not carry any glycosyl substitution. The deacylated glycolipid fraction contained the free kojibiose-Gro oligosaccharide, identical to the backbone of the GL anchor of LTA, and its shorter fragment α-Glc-1-Gro. These OS may have originated from the GL precursors of LTA biosynthesis.
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Glucolípidos , Lactococcus lactis , Ácidos Teicoicos/química , Glicerol , Lactococcus lactis/química , Lipopolisacáridos/química , FosfatosRESUMEN
Floricoccus penangensis is a Gram-positive coccoid organism that is a member of the lactic acid bacteria. F. penangensis ML061-4 was originally isolated from the surface of an Assam tea leaf, and its genome is herein shown to contain gene clusters predicted to be involved in complex carbohydrate metabolism and biosynthesis of secondary metabolites.
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Bacteriophages (or phages) represent a persistent threat to the success and reliability of food fermentation processes. Recent reports of phages that infect Streptococcus thermophilus have highlighted the diversification of phages of this species. Phages of S. thermophilus typically exhibit a narrow range, a feature that is suggestive of diverse receptor moieties being presented on the cell surface of the host. Cell wall polysaccharides, including rhamnose-glucose polysaccharides and exopolysaccharides have been implicated as being involved in the initial interactions with several phages of this species. Following internalization of the phage genome, the host presents several defences, including CRISPR-Cas and restriction and modification systems to limit phage proliferation. This review provides a current and holistic view of the interactions of phages and their S. thermophilus host cells and how this has influenced the diversity and evolution of both entities.
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Bacteriófagos , Fagos de Streptococcus , Bacteriófagos/genética , Streptococcus thermophilus , Reproducibilidad de los Resultados , Polisacáridos/metabolismoRESUMEN
Lactococcus lactis and Lactococcus cremoris are Gram-positive lactic acid bacteria widely used as starter in milk fermentations. Lactococcal cells are covered with a polysaccharide pellicle (PSP) that was previously shown to act as the receptor for numerous bacteriophages of the Caudoviricetes class. Thus, mutant strains lacking PSP are phage resistant. However, because PSP is a key cell wall component, PSP-negative mutants exhibit dramatic alterations of cell shape and severe growth defects, which limit their technological value. In the present study, we isolated spontaneous mutants with improved growth, from L. cremoris PSP-negative mutants. These mutants grow at rates similar to the wild-type strain, and based on transmission electron microscopy analysis, they exhibit improved cell morphology compared to their parental PSP-negative mutants. In addition, the selected mutants maintain their phage resistance. Whole-genome sequencing of several such mutants showed that they carried a mutation in pbp2b, a gene encoding a penicillin-binding protein involved in peptidoglycan biosynthesis. Our results indicate that lowering or turning off PBP2b activity suppresses the requirement for PSP and ameliorates substantially bacterial fitness and morphology. IMPORTANCE Lactococcus lactis and Lactococcus cremoris are widely used in the dairy industry as a starter culture. As such, they are consistently challenged by bacteriophage infections which may result in reduced or failed milk acidification with associated economic losses. Bacteriophage infection starts with the recognition of a receptor at the cell surface, which was shown to be a cell wall polysaccharide (the polysaccharide pellicle [PSP]) for the majority of lactococcal phages. Lactococcal mutants devoid of PSP exhibit phage resistance but also reduced fitness, since their morphology and division are severely impaired. Here, we isolated spontaneous, food-grade non-PSP-producing L. cremoris mutants resistant to bacteriophage infection with a restored fitness. This study provides an approach to isolate non-GMO phage-resistant L. cremoris and L. lactis strains, which can be applied to strains with technological functionalities. Also, our results highlight for the first time the link between peptidoglycan and cell wall polysaccharide biosynthesis.